Corrinoid-dependent enzymes are widespread in nature and play key roles in human, animal and microbial metabolism. Two known vitamin B12-dependent enzymes exist in humans: methylcobalamin (MeCbl)-dependent methionine synthase and adenosylcobalamin (AdoCbl)-dependent methylmalonyl-coenzyme A mutase. The clinical hallmarks of a vitamin B12 deficiency are megaloblastic anemia (‘pernicious anemia’) and/or neuropathies. The B12-dependent enzyme reactions play a vital role in maintaining healthy nerve and red blood cells and are required for the synthesis of DNA. Formula I gives the structure of the two coenzyme forms of vitamin B12 and related B12 derivatives (cobalamins) which have been isolated from human cells, blood or tissue. The α (or lower) axial site is occupied by an intramolecularly-bound 5,6-dimethylbenzimidazole, and the β (or upper) axial site can be occupied by a variety of ligands.
Upon reaching cells, cobalamin derivatives are converted to MeCbl (III) and AdoCbl (IV) by currently ill-defined mechanisms. Many studies have been carried out over the past several decades involving the extraction and identification of cobalamins from mammalian cells, tissue and blood, in addition to other biological samples such as foods and seaweed. AdoCbl, MeCbl and aquacobalamin (II, H2OCbl+⇄HOCbl+H+; pKa=7.8) are the major cobalamin metabolites isolated from biological samples (Jacobsen, D. W., et al, Method. Enzymol., 1986, 123, 14-22). Sulfitocobalamin (SO3Cbl−, VI) is also isolable from mammalian cells and foods (see Jacobsen et al, above), and there are also reports of the isolation of nitrocobalamin (NO2Cbl, VII) from biological sources (Anes, J. M., et al, J. Chromatogr. B Biomed. Appl., 1994, 660, 180-85). Whether or not cyanocobalamin (I) is truly “naturally occurring” is controversial; some studies report small amounts of this derivative, especially in smokers (Koyama, K., et al, Nephrol. Dial. Transplant., 1997, 12, 1622-28).
Thiol derivatives of B12, thiolatocobalamins (X=thiol, Formula I) were first identified in the 1960s, but have not attracted much attention until recently. Glutathionylcobalamin (GSCbl, V) is a naturally occurring intracellular form of cobalamin. Formation of GSCbl from H2OCbl+ and glutathione is irreversible (Kobs˜3×108 M−1 at pH 7.4, 25° C.) and rapid (t½˜3 s for [GSH]=5 mM, pH 7.4, 37° C.) (Xia, L., et al, Inorg Chem., 2004, 43, 6848-57). This compound is an important cobalamin metabolite in mammals; it is more readily absorbed and retained longer than cyanocobalamin, and is more active than other cobalamins in promoting methionine synthase activity in rabbit spleen extracts (Pezacka, E., et al, Biochem. Biophys. Res. Comm., 1990, 169, 443-50). It has been proposed that, in vivo, GSCbl is an intermediate in the conversion of biologically inactive cobalamin forms to the active coenzyme forms adenosylcobalamin (IV) and methylcobalamin (III) (see Pezacka et al, above). The reducing agent glutathione (GSH) is required for the formation of GSCbl, and is present in lower levels under oxidative stress conditions. An alternative role for GSCbl was also recently proposed, in which the formation of GSCbl prevents B12 from being scavenged by xenobiotics (Watson, W. P., et al, Chem. Res. Toxicol., 2004, 17, 1562-67). In addition, McCaddon and co-workers suggested that GSCbl and related thiolatocobalamins might be more effective than currently available pharmaceutical B12 forms (CNCbl and H2OCbl+) in treating of B12-related conditions associated with oxidative stress such as Alzheimer's disease (McCaddon, A., et al, Neurology, 2002, 58, 1395-99). Thus, GSCbl and other thiolatocobalamins have the potential to offer a valuable source of cobalamin in therapeutic applications requiring administration of a vitamin B12 derivative.
The structure of the thiol N-acetyl-L-cysteine (NAC) is shown in Formula II. NAC has been used in the clinic for over 50 years as a mucolytic agent. NAC also promotes the synthesis of GSH, is an antioxidant, and may be a useful therapeutic for treatment of oxidative stress-linked
diseases (Aitio, M. Br. J. Clin. Pharm., 2005, 61, 5-15).
The present invention relates to the synthesis of the novel vitamin B12 derivative N-acetyl-L-cysteinylcobalamin, Na[NACCbl]. Na[NACCbl] is characterized by a variety of techniques, including 1H NMR and UV-vis spectroscopies and mass spectrometry. The structure of Na[NACCbl] is further verified by X-ray crystallography, and an XAS spectrum has been recorded. Na[NACCbl]) is of interest, given that it has been shown that co-administrating H2OCbl+ and N-acetyl-L-cysteine improves cognitive performance in Alzheimer's patients, and the possibility that NACCbl−, rather than the individual compounds themselves, is responsible for the beneficial effects of this approach.
The prior art describes a method of preparing GSCbl by reacting a 1:1 ratio hydroxocobalamin (=hydroxycobalamin or aquacobalamin) and glutathione in water and precipitating the resulting complex to give a product of at least 95% purity (GB 945722 (1964 to Merck & Co., Inc.)). However, the technical sophistication available today for determining product purity was not available at the time of this earlier work and subsequent repeated attempts of this work have identified that the resulting product is in fact of only 60-70% purity. More recently, other methods for preparing GSCbl in high purity have been reported (Pezacka, E., et al, Biochem. Biophys. Res. Commun., 1990, 169, 443-50; Brown K., et al, Biochem., 1993, 32, 8421-28; Brasch N., et al, Inorg. Chem., 1999, 76, 197-209) using a large excess of glutathione (5-12×); however, an additional chromatographic step is required to provide a product of 98% purity. One of the present inventors previously developed procedures for synthesizing and isolating the thiolatocobalamins γ-glutamylcysteinylcobalamin and glutathionylcobalamin in aqueous solution in high yield and purity by the addition of a small excess of thiol to a highly concentrated solution of aquacobalamin, followed by the addition of acetone to precipitate the product after completion of the reaction (Suto, R. K., et al, Inorg. Chem., 2001, 40, 2686-92; U.S. Pat. No. 7,030,105). This patent presented a method of preparing GSCbl of an acceptable purity level which does not require the cost and effort of a chromatographic purification step. It is performed in the presence of air. A similar procedure was also used to synthesize pentafluorophenylthiolatocobalamin and cyclohexylthiolatocobalamin in methanol under anaerobic conditions (Brasch, N. E., et al, J. Inorg. Biochem., 1999, 76, 197-209; Hsu, T.-L. C., et al, Inorg. Chem., 1998, 37, 5109-5116).